Best Practices for Differential Scanning Calorimetry of Proteins and Biopolymers

por Verna Frasca, Tuesday, 14th August 2018

iStock_000018432116_300x270In a previous blog, I discussed best practices to achieve high-quality data with a MicroCal DSC. Here are more best practices for the MicroCal PEAQ-DSC system, as well as the MicroCal VP-Capillary DSC and VP-DSC systems. These best practices apply to the analysis of proteins in solution, and other biopolymers.

 

 

Sample requirements:

  • For high-quality Differential Scanning Calorimetry (DSC) data, the samples need to be in desired buffer (pH, salt, excipients, etc.), and the matched buffer is used in the DSC reference cell and for buffer-buffer scans.
  • Prepare samples in the desired buffer by dialysis, a desalting column, or another buffer exchange method.
  • Save the buffer from dialysis/buffer exchange to use for the reference cell, buffer-buffer scans, and sample dilution.
  • Recommended protein concentration range of 0.1 mg/ml to 2 mg/ml.
  • Tms from DSC can be concentration-dependent.
    • For Tm screening and comparability/similarity studies, be sure all proteins are matched in concentration.

DSC settings:

  • When using DSC for Tm screening and comparability/similarity studies, be sure all settings are the same, including scan rate, temperature range, feedback mode, pre-scan thermostat.
    • Tms and DSC thermograms can be dependent on scan rate and feedback mode.
  • Temperature range: start scan 10-20°C below 1st Tm, and end scan 10-20°C above final Tm.
  • Scan rates:
    • Typical scan rate for proteins and nucleic acids is 60 to 90 °C/h
    • Typical scan rate for lipids is 30 to 60°C/h
    • For MicroCal PEAQ-DSC and VP-Capillary DSC, use scan rate of 180 to 240 °C/h to increase sample throughput during Tm screening
  • Recommended “Feedback Modes”:
    • proteins – none or low
    • DNA and RNA – low or medium
    • Lipids – high
  • Recommended “Pre-scan thermostat” of 3 to 15 minutes.

For automated MicroCal PEAQ-DSC and VP-Capillary DSC systems:

  • Be sure the samples are stored at an appropriate temperature prior to DSC analysis. For proteins and other biomolecules, set the cooling stack temperature to 5 to 10°C. If you are studying a thermostable material, you can set the cooling stack to 25°C.
  • Use a minimum of three purge refills (called “number of filling strokes” on the VP-Capillary DSC) to fill the DSC cells from the 96 well plate.
  • Program cell cleanings using 20% Contrad 70 or 14% Decon 90.

DSC raw data quality:

  • Always have a matched buffer-buffer scan (run under identical scan conditions) to use for data analysis. If desired, you can perform a buffer-buffer scan prior to each sample.
  • Frequent buffer-buffer scans serve as an additional instrument performance check to make sure the DSC cells are clean, and the instrument is functioning.
  • Buffer-buffer scans should be reproducible throughout the sequence (assuming the same DSC run parameters).
  • With the Automated PEAQ-DSC, and its efficient wash pump and optimized DSC cell cleaning protocols, it is possible to run three (or more) samples in a row, without running buffer-buffer scans in-between.
  • The DP value (in mcal/min) for the raw DSC thermogram should be close to zero at the start of buffer-buffer or water-water scans.
  • At the start of the protein scans, the DP value typically becomes more negative, compared to matched buffer-buffer scans. Higher protein concentrations have a larger negative deflection. With good buffer matching, this DP difference should be approximately 0.15 to 0.25 mcal/min for every 1 g/L protein (Figure 1).
  • If the DP difference between protein and buffer-buffer scans is larger than 1 mcal/min, this can be due to a buffer mismatch between protein and buffer, a dirty cell, an underfilled cell, bubbles in cell, or more concentrated protein (Figure 2).

DSC best practices Figure 1

Figure 1.

DSC best practices Figure 2

Figure 2.

Watch this video explaining What is Differential Scanning Calorimetry

Related content: